Method and Apparatus for Detecting Nucleotides

ABSTRACT

A system and method employing at least one semiconductor device, or an arrangement of insulating and metal layers, having at least one detecting region which can include, for example, a recess or opening therein, for detecting a charge representative of a component of a polymer, such as a nucleic acid strand proximate to the detecting region, and a method for manufacturing such a semiconductor device. The system and method can thus be used for sequencing individual nucleotides or bases of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The semiconductor device includes at least two doped regions, such as two n-typed regions implanted in a p-typed semiconductor layer or two p-typed regions implanted in an n-typed semiconductor layer. The detecting region permits a current to pass between the two doped regions in response to the presence of the component of the polymer, such as a base of a DNA or RNA strand. The current has characteristics representative of the component of the polymer, such as characteristics representative of the detected base of the DNA or RNA strand.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.13/617,626, filed on Sep. 14, 2012, which is a continuation of U.S.patent application Ser. No. 13/409,800, filed on Mar. 1, 2012, now U.S.Pat. No. 8,426,232, which is a continuation of U.S. patent applicationSer. No. 11/301,259, filed on Dec. 13, 2005, now U.S. Pat. No.8,232,582, which is a division of U.S. patent application Ser. No.10/258,439, filed on Oct. 24, 2002, now U.S. Pat. No. 7,001,792, whichis the U.S. national stage of International Patent Application No.PCT/US01/13101, filed on Apr. 24, 2001. International Patent ApplicationNo. PCT/US01/13101 is a continuation-in-part of U.S. patent applicationSer. No. 09/653,543, filed on Aug. 31, 200, now U.S. Pat. No. 6,413,792,and claims the benefit of U.S. Provisional Patent Application Ser. No.60/259,584, filed on Jan. 4, 2001, of U.S. Provisional PatentApplication Ser. No. 60/199,130, filed on Apr. 24, 2000, and of U.S.Provisional Patent Application Ser. No. 60/217,681, filed on Jul. 12,2000. U.S. patent application Ser. No. 09/653,543 claims the benefit ofU.S. Provisional Patent Application Ser. No. 60/199,130, filed on Apr.24, 2000, and of U.S. Provisional Patent Application Ser. No.60/217,681, filed on Jul. 12, 2000. The entire contents of all of saidprior applications are incorporated herein by reference.

BACKGROUND

1. Field of the Disclosure

The present invention relates to a system and method employing asemiconductor device having a detecting region for identifying theindividual mers of long-chain polymers, such as carbohydrates andproteins, as well as individual bases of deoxyribonucleic acid (DNA) orribonucleic acid (RNA), and a method for making the semiconductordevice. More particularly, the present invention relates to a system andmethod employing a semiconductor device, similar to a field-effecttransistor device, capable of identifying the bases of a DNA/RNA strandto thus enable sequencing of the strand to be performed.

2. Description of the Related Art

DNA consists of two very long, helical polynucleotide chains coiledaround a common axis. The two strands of the double helix run inopposite directions. The two strands are held together by hydrogen bondsbetween pairs of bases, consisting of adenine (A), thymine (T), guanine(G), and cytosine (C). Adenine is always paired with thymine, andguanine is always paired with cytosine. Hence, one strand of a doublehelix is the complement of the other.

Genetic information is encoded in the precise sequence of bases along aDNA strand. In normal cells, genetic information is passed from DNA toRNA. Most RNA molecules are single stranded but many contain extensivedouble helical regions that arise from the folding of the chain intohairpin-like structures.

Mapping the DNA sequence is part of a new era of genetic-based medicineembodied by the Human Genome Project. Through the efforts of thisproject, one day doctors will be able to tailor treatment to individualsbased upon their genetic composition, and possibly even correct geneticflaws before birth. However, to accomplish this task it will benecessary to sequence each individual's DNA. Although the human genomesequence variation is approximately 0.1%, this small variation iscritical to understanding a person's predisposition to various ailments.In the near future, it is conceivable that medicine will be “DNApersonalized,” and a physician will order sequence information just asreadily as a cholesterol test is ordered today. Thus, to allow suchadvances to be in used in everyday life, a faster and more economicalmethod of DNA sequencing is needed.

One method of performing DNA sequencing is disclosed in U.S. Pat. No.5,653,939, the entire content of which is incorporated herein byreference. This method employs a monolithic array of test sites formedon a substrate, such as a semiconductor substrate. Each test siteincludes probes which are adapted to bond with a predetermined targetmolecular structure. The bonding of a molecular structure to the probeat a test site changes the electrical, mechanical and optical propertiesof the test site. Therefore, when a signal is applied to the test sites,the electrical, mechanical, or optical properties of each test site canbe measured to determine which probes have bonded with their respectivetarget molecular structure. However, this method is disadvantageousbecause the array of test sites is complicated to manufacture, andrequires the use of multiple probes for detecting different types oftarget molecular structures.

Another method of sequencing is known as gel electrophoresis. In thistechnology, the DNA is stripped down to a single strand and exposed to achemical that destroys one of the four nucleotides, for example A, thusproducing a strand that has a random distribution of DNA fragmentsending in A and labeled at the opposite end. The same procedure isrepeated for the other three remaining bases. The DNA fragments areseparated by gel electrophoresis according to length. The lengths showthe distances from the labeled end to the known bases, and if there areno gaps in coverage, the original DNA strand fragment sequence isdetermined.

This method of DNA sequencing has many drawbacks associated with it.This technique only allows readings of approximately 500 bases, since aDNA strand containing more bases would “ball” up and not be able to beread properly. Also, as strand length increases, the resolution in thelength determination decreases rapidly, which also limits analysis ofstrands to a length of 500 bases. In addition, gel electrophoresis isvery slow and not a workable solution for the task of sequencing thegenomes of complex organisms. Furthermore, the preparation before andanalysis following electrophoresis is inherently expensive and timeconsuming. Therefore, a need exists for a faster, consistent and moreeconomical means for DNA sequencing.

Another approach for sequencing DNA is described in U.S. Pat. Nos.5,795,782 and 6,015,714, the entire contents of which are incorporatedherein by reference. In this technique, two pools of liquid areseparated by a biological membrane with an alpha hemolysin pore. As theDNA traverses the membrane, an ionic current through the pore isblocked. Experiments have shown that the length of time during which theionic current through the pore is blocked is proportional to the lengthof the DNA fragment. In addition, the amount of blockage and thevelocity depend upon which bases are in the narrowest portion of thepore. Thus, there is the potential to determine the base sequence fromthese phenomena.

Among the problems with this technique are that individual nucleotidescannot, as yet, be distinguished. Also, the spatial orientation of theindividual nucleotides is difficult to discern. Further, the electrodesmeasuring the charge flow are a considerable distance from the pore,which adversely affects the accuracy of the measurements. This islargely because of the inherent capacitance of the current-sensingelectrodes and the large statistical variation in sensing the smallamounts of current. Furthermore, the inherent shot noise and other noisesources distort the signal, incurring additional error. Therefore, aneed exists for a more sensitive detection system which discriminatesamong the bases as they pass through the sequencer.

SUMMARY

An object of the present invention is to provide a system and method foraccurately and effectively identifying individual bases of DNA or RNA.

Another object of the present invention is to provide a system andmethod employing a semiconductor device for sequencing individual basesof DNA or RNA.

A further object of the present invention is to provide a method formanufacturing a semiconductor-based DNA or RNA sequencing device.

Another object of the present invention is to provide a system andmethod for accurately and effectively identifying the individual mers oflong-chain polymers, such as carbohydrates or proteins, as well asmeasuring the lengths of the long-chain polymers.

Still another object of the present invention is to provide a system andmethod employing a semiconductor-based device having an opening therein,for accurately and effectively identifying bases of DNA or RNA bymeasuring charge at a location where the DNA or RNA molecules traversethe opening in the sequencer, to thus eliminate or at least minimize theeffects of shot noise and other noise sources associated with the randommovement of the DNA or RNA molecules through the opening.

These and other objects of the invention are substantially achieved byproviding a system for detecting at least one polymer, comprising atleast one semiconductor device, or an arrangement of insulating andmetal layers, having at least one detecting region which is adapted todetect a charge representative of a component of the polymer proximateto the detecting region. The component can include a base in a nucleicacid strand, so that the detecting region is adapted to detect thecharge which is representative of the base in the nucleic acid strand.The detecting region is further adapted to generate a signalrepresentative of the detected charge. Also, the detecting region caninclude a region of the semiconductor device defining a recess in thesemiconductor device, or an opening in the semiconductor device having across-section sufficient to enable the polymer to enter the opening, sothat the detecting region detects the charge of the component in theopening. Furthermore, the semiconductor device preferably furtherincludes at least two doped regions, and the detecting region can pass acurrent between the two doped regions in response to a presence of thecomponent proximate to the detecting region.

The above and other objects of the invention are also substantiallyachieved by providing a method for detecting at least one polymer,comprising the steps of positioning a portion of the polymer proximateto a detecting region of at least one semiconductor device, anddetecting at the detecting region a charge representative of a componentof the polymer proximate to the detecting region. The component caninclude a base in a nucleic acid strand, so that the detecting stepdetects a charge representative of the base. The method furthercomprises the step of generating at the detecting region a signalrepresentative of the detected charge. The detecting region can includea region of the semiconductor device defining a recess in thesemiconductor device, or an opening in the semiconductor device having across-section sufficient to enable the polymer to enter the opening, sothat the detecting step detects the charge of the component in therecess or opening. Furthermore, the semiconductor device can furtherinclude at least two doped regions, so that the method can furtherinclude the step of passing a current between the two doped regions inresponse to a presence of the component proximate to the detectingregion.

The above and other objects of the invention are further substantiallyachieved by providing a method for manufacturing a device for detectinga polymer, comprising the steps of providing a semiconductor structurecomprising at least one semiconductor layer, and creating a detectingregion in the semiconductor structure, such that the detecting region isadapted to detect a charge representative of a component of the polymerproximate to the detecting region. The component can include a base in anucleic acid strand, and the detecting region can be created to detect acharge representative of the base in the nucleic acid strand. The methodcan further include the step of creating a recess in the semiconductorstructure, or creating an opening in the semiconductor structure havinga cross-section sufficient to enable a portion of the polymer to passtherethrough, and being positioned in relation to the detecting regionsuch that the detecting region is adapted to detect the chargerepresentative of the component in the recess or opening. The method canfurther include the step of forming an insulating layer on a wall of thesemiconductor layer having the opening to decrease the cross-section ofthe opening. Furthermore, the method can include the step of creating atleast two doped regions in the semiconductor layer which are positionedwith respect to the detecting region such that the detecting region isadapted to pass a current between the doped regions in response to thecomponent of the polymer proximate to the detecting region. The dopedregions can be separated by a portion of the semiconductor layer havinga different doping, and can be created as a stack of doped regions, eachhaving a first doping and being separated by a layer having a seconddoping. The doped regions can include either a p-type or an n-typedoping.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, advantages and novel features of the inventionwill be more readily appreciated from the following detailed descriptionwhen read in conjunction with the accompanying drawings, in which:

FIG. 1 illustrates a system for performing DNA or RNA sequencingcomprising a DNA or RNA sequencer constructed in accordance with anembodiment of the present invention;

FIG. 2 illustrates a top view of the DNA or RNA sequencer shown in FIG.1;

FIG. 3 is a graph showing an example of the waveform, representing thecurrent detected by a current detector in the system shown in FIG. 1 asthe adenine (A), thymine (T), guanine (G), and cytosine (C) bases of aDNA or RNA sequence pass through the DNA or RNA sequencer;

FIG. 4 illustrates a cross-sectional view of a silicon-on-insulator(SOI) substrate from which a DNA or RNA sequencer as shown in FIG. 1 isfabricated in accordance with an embodiment of the present invention;

FIG. 5 illustrates a cross-sectional view of the SOI substrate shown inFIG. 5 having shallow and deep n-type regions formed in the siliconlayer, and a portion of the substrate etched away;

FIG. 6 illustrates a cross-sectional view of the SOI substrate shown inFIG. 5 in which a portion of the insulator has been etched away andanother shallow n-type region has been formed in the silicon layer;

FIG. 7 illustrates a cross-sectional view of the SOI substrate having anopening etched therethrough;

FIG. 8 illustrates a top view of the SOI substrate as shown in FIG. 7;

FIG. 9 illustrates a cross-sectional view of the SOI substrate shown inFIG. 7 having an oxidation layer formed on the silicon layer and on thewalls forming the opening therein;

FIG. 10 illustrates a top view of the SOI substrate as shown in FIG. 9;

FIG. 11 illustrates a detailed cross-sectional view of the SOI substrateshown in FIG. 7 having an oxidation layer formed on the silicon layerand on the walls forming the opening therein;

FIG. 12 illustrates a top view of the SOI substrate shown in FIG. 11;

FIG. 13 illustrates a detailed cross-sectional view of an exemplaryconfiguration of the opening in SOI substrate shown in FIG. 7;

FIG. 14 illustrates a top view of the opening shown in FIG. 13;

FIG. 15 illustrates a cross-sectional view of the SOI substrate as shownin FIG. 9 having holes etched in the oxidation layer and metal contactsformed over the holes to contact the shallow and deep n-type regions,respectively;

FIG. 16 illustrates a cross-sectional view of the DNA or RNA sequencershown in FIG. 1 having been fabricated in accordance with themanufacturing steps shown in FIGS. 4-15;

FIG. 17 illustrates a top view of a DNA or RNA sequencer having multipledetectors formed by multiple n-type regions according to anotherembodiment of the present invention;

FIG. 18 illustrates a cross-sectional view of a DNA or RNA sequenceraccording to another embodiment of the present invention;

FIG. 19 illustrates a cross-sectional view of a DNA or RNA sequenceraccording to a further embodiment of the present invention;

FIG. 20 illustrates a cross-sectional view of a DNA or RNA sequenceraccording to a further embodiment of the present invention;

FIG. 21 illustrates a top view of the DNA or RNA sequencer shown in FIG.20;

FIG. 22 is a conceptual block diagram illustrating an example of amatrix arrangement of DNA or RNA sequencers;

FIG. 23 is a cross-sectional view of a sequencer having a metal layer inplace of a middle semiconductor layer to achieve electron tunneling;

FIG. 24 illustrates a detailed cross-sectional view of another exemplaryconfiguration of the opening in SOI substrate shown in FIG. 7;

FIG. 25 illustrates a top view of the opening shown in FIG. 24;

FIG. 26 illustrates a cross-sectional view of a multi-opening sequencerused with separate liquid regions;

FIGS. 27A and 27B are images of photographs of opening patterns formedin a semiconductor structure; and

FIGS. 28A and 28B are images of photographs of opening patterns forms ina semiconductor structure.

DETAILED DESCRIPTION

FIGS. 1 and 2 illustrate a system 100 for detecting the presence of apolymer, such as DNA or RNA, a protein or carbohydrate, or a long chainpolymer such as petroleum, and more preferably, for identifying theindividual mers of the polymer or long chain polymer, as well as thelength of the polymer or long chain polymer. The system 100 ispreferably adaptable for performing sequencing of nucleic acids, such asDNA or RNA sequencing, according to an embodiment of the presentinvention. Accordingly, for purposes of this description, the system 100will be discussed in relation to nucleic acid sequencing.

The system 100 includes a nucleic acid sequencing device 102 which, asdescribed in more detail below, is a semiconductor device. Specifically,the nucleic acid sequencing device 102 resembles a field-effecttransistor, such as a MOSFET, in that it includes two doped regions, adrain region 104 and a source region 106. However, unlike a MOSFET, thenucleic acid sequencing device does not include a gate region forreasons discussed below.

The nucleic acid sequencing device 102 is disposed in a container 108that includes a liquid 110 such as water, gel, a buffer solution such asKCL, or any other suitable solution. It is important to note that thesolution 110 can be an insulating medium, such as oil, or any othersuitable insulating medium. In addition, the container 108 does not needto include a medium such as a liquid. Rather, the container 108 can besealed and evacuated to create a vacuum in which nucleic acid sequencingdevice 102 is disposed. Also, although FIG. 1 shows only a singlenucleic acid sequencing device 102 in the container 108 for exemplarypurposes, the container can include multiple nucleic acid sequencingdevices 102 for performing multiple DNA sequencing measurements inparallel.

The liquid 110 or other medium or vacuum in container 108 includes thenucleic acid strands or portions of nucleic acid strands 111 to besequenced by nucleic acid sequencing device 102. As further shown,voltage source 112, such as a direct current voltage source, is coupledin series with a current meter 114 by leads 116 across drain and sourceregions 104 and 106, respectively. In this example, the positive lead ofvoltage source 112 is coupled to the drain region 104 while the negativelead of voltage source 112 is coupled via the current meter 114 tosource region 106.

The voltage potential applied across drain and source regions 104 and106 of nucleic acid sequencing device 102 can be small, for example,about 100 mV, which is sufficient to create a gradient across drain andsource regions 104 and 106, to draw the nucleic acid strands intoopening 118 of the nucleic acid sequencing device 102. That is, thenucleic acid strands 111 move through the opening 118 because of thelocal gradient. Alternatively or in addition, the liquid can include anionic solution. In this event, the local gradient causes the ions in thesolution to flow through the opening 118, which assists the nucleic acidstrands 111, such as DNA or RNA, to move through the opening 118 aswell.

Additional electrodes 113 and 115 positioned in the medium 110 andconnected to additional voltage sources 117 and 121 would furtherfacilitate the movement of the nucleic acid strands towards the opening118. In other words, the external electrodes 113 and 115 are used toapply an electric field within the medium 110. This field causes all ofthe charged particles, including the nucleic acid strand 111, to floweither toward the opening 118 or away from the opening 118. Thuselectrodes 113 and 115 are used as a means to steer the nucleic acidstrands 111 into or out of the opening 118. In order to connect voltagesources 112 and 117 to the nucleic acid sequencer 102, metal contacts123 are coupled to the n-type doped region 128 and 130, described inmore detail below. The electrodes 113 and 115 could also provide a highfrequency voltage which is superimposed on the DC voltage by analternating voltage source 125. This high frequency voltage, which canhave a frequency in the radio frequency range, such as the megahertzrange (e.g., 10 MHz), causes the nucleic acid strand 111 and ions tooscillate. This oscillation makes passage of the nucleic acid strand 111through the opening 118 smoother, in a manner similar to shaking a saltshaker to enable the salt grains to pass through the openings in theshaker. Alternatively, a device 127, such as an acoustic wave generator,can be disposed in the liquid 110 or at any other suitable location, andis controlled to send sonic vibrations through the device 102 to providea similar mechanical shaking function.

As can be appreciated by one skilled in the art, the nucleic acidstrands each include different combinations of bases A, C, G and T,which each contain a particular magnitude and polarity of ionic charge.The charge gradient between drain and source regions 104 and 106, orotherwise across the opening 118, will thus cause the charged nucleicacid strands to traverse the opening 118. Alternatively, another voltagesource (not shown) can be used to create a difference in voltagepotential between the opening 118 and the liquid. Also, a pressuredifferential can be applied across the opening 118 to control the flowof the DNA independent from the voltage applied between the drain andsource 104 and 106.

In addition, the Sequencing device 102 can attract the nucleic acidstrands to the opening 118 by applying a positive voltage to the medium110 relative to the voltage source. Furthermore, the nucleic acidstrands in the medium 110 can be pushed in and out of the opening 118and be analyzed multiple times by reversing the polarity across drainand source regions 104 and 106, respectively.

As described in more detail below, the opening 118 is configured to havea diameter within the nanometer range, for example, within the range ofabout 1 nm to about 10 nm. Therefore, only one DNA strand can passthrough opening 118 at any given time. As a DNA strand passes throughopening 118, the sequence of bases induce image charges which form achannel 119 between the drain and source regions 104 and 106 thatextends vertically along the walls of the device defining opening 118.As a voltage is applied between the source 136 and drain 128 by means ofthe voltage source 112, these image charges in the channel flow fromsource to drain, resulting in a current flow which can be detected bythe current meter 114. The current exists in the channel as long as thecharge is present in the opening 118, and thus the device currentdetected by the current meter 114 is much larger than the currentassociated with the moving charge. For example, a singly charged ionpassing through the opening 118 in one microsecond accounts for an ioncurrent of 0.16 pA and a device current of 160 nA.

Alternatively, the bases induce a charge variation in channel 119,leading to a current variation as detected by current meter 114. Anyvariation of the ion flow through the opening due to the presence of theDNA strand would also cause a variation to the image charge in thechannel 119 and results in a current variation as detected by currentmeter 114. That is, the device current measured by current meter 114will diminish from, for example, 80.mu.A to 4.mu.A. as the DNA strand111 passes through opening 118.

Each different type of bases A, C, G, and T induces a current having aparticular magnitude and waveform representative of the particularcharge associated with its respective type of bases. In other words, anA type base will induce a current in a channel between the drain andsource regions of the nucleic acid sequencing device 102 having amagnitude and waveform indicative of the A type base. Similarly, the C,T and G bases will each induce a current having a particular magnitudeand waveform.

An example of a waveform of the detected current is shown in FIG. 3,which symbolically illustrates the shape, magnitude, and time resolutionof the expected signals generated by the presence of the A, C, G and Tbases. The magnitude of current is typically in the microampere (.mu.A)range, which is a multiplication factor of 10.sup.6 greater than the ioncurrent flowing through the opening 118, which is in the picoampererange. A calculation of the electrostatic potential of the individualbases shows the complementary distribution of charges that lead to thehydrogen bonding. For example, the T-A and C-G pairs have similardistributions when paired viewed from the outside, but, when unpaired,as would be the case when analyzing single-stranded DNA, the surfaceswhere the hydrogen bonding occurs are distinctive. The larger A and Gbases are roughly complementary (positive and negative reversed) on thehydrogen bonding surface with similar behavior for the smaller T and Cbases.

Accordingly, as the DNA strand passes through opening 118, the sequenceof bases in the strand can be detected and thus ascertained byinterpreting the waveform and magnitude of the induced current detectedby current meter 114. The system 100 therefore enables DNA sequencing tobe performed in a very accurate and efficient manner.

Since the velocity of the electrons in the channel 119 is much largerthan the velocity of the ions passing through the opening, the draincurrent is also much larger than the ion current through the opening118. For an ion velocity of 1 cm/s and an electron velocity of 10.sup.6cm/s, an amplification of 1 million can be obtained.

Also, the presence of a DNA molecule can be detected by monitoring thecurrent I.sub.p through the opening 118. That is, the current I.sub.pthrough the opening reduces from 80 pA to 4 pA when a DNA moleculepasses through the opening. This corresponds to 25 electronic chargesper microsecond as the molecule passes through the opening.

Measurement of the device current rather than the current through theopening has the following advantages. The device current is much largerand therefore easier to measure. The larger current allows an accuratemeasurement over a short time interval, thereby measuring the chargeassociated with a single DNA base located between the two n-typeregions. In comparison, the measurement of the current through theopening has a limited bandwidth, limited by the shot-noise associatedwith the random movement of charge through the opening 118. For example,measuring a 1 pA current with a bandwidth of 10 MHz yields an equivalentnoise current of 3.2 pA. Also, the device current can be measured evenif the liquids on both sides of the opening 118 are not electricallyisolated. That is, as discussed above, the sequencing device 102 isimmersed in a single container of liquid. Multiple sequencers 102 canthus be immersed in a single container of liquid to enable multiplecurrent measurements to be performed in parallel. Furthermore, thenanometer-sized opening 118 can be replaced by any other structure ormethod which brings the DNA molecule in close proximity to the twon-type regions, as discussed in more detail below.

The preferred method of fabricating a nucleic acid sequencing device 102will now be described with reference to FIGS. 4-16. As shown in FIG. 4,the fabrication process begins with a wafer 120, such as asilicon-on-insulator (SOI) substrate comprising a silicon substrate 122,a silicon dioxide (SiO.sub.2) layer 124, and a thin layer of p-typesilicon 126. In this example, the silicon substrate 122 has a thicknesswithin the range of about 300.mu.m to about 600.mu.m, the silicondioxide layer 124 has a thickness within the range of about 200 to 6400nm, and the p-type silicon layer 126 has a thickness of about 1.mu.m orless (e.g., within a range of about 10 nm to about 1000 nm).

As shown in FIG. 5, a doped n-type region 128 is created in the p-typesilicon layer 126 by ion implantation, and annealing or diffusion of ann-type dopant, such as arsenic, phosphorous or the like. As illustrated,the n-type region 128 is a shallow region which does not pass entirelythrough p-type silicon 126. A deep n-type region 130 is also created inthe p-type silicon 126 as illustrated in FIG. 5. The deep n-type region130 passes all the way through the p-type silicon 126 to silicon dioxide124 and is created by known methods, such as diffusion, or ionimplantation and annealing of an n-type material which can be identicalor similar to the n-type material used to create n-type region 128. Asfurther illustrated in FIG. 5, the silicon substrate 122 is etched alongits (111) plane by known etching methods, such as etching in potassiumhydroxide (KOH) or the like. The back of the substrate 122 can also beetched with a teflon jig. As illustrated, the etching process etchesaway a central portion of silicon substrate 122 down to the silicondioxide 124 to create an opening 132 in the silicon substrate 122.

As shown in FIG. 6, the portion of the silicon dioxide 124 exposed inopening 132 is etched away by conventional etching methods, such asetching in hydrofluoric acid, reactive etching or the like. Anothershallow n-type region 124 is created in the area of the p-type silicon126 exposed at opening 132 by known methods, such implantation ordiffusion of an n-type material identical or similar to those used tocreate n-type regions 128 and 130.

Opening 118 (see FIGS. 1 and 2) is then formed through the n-type region128, p-type silicon 126 and bottom n-type region 134 as shown, forexample, in FIGS. 7 and 8 by reactive ion etching (RIE) using Freon 14(CF.sub.4), optical lithography, electron-beam lithography or any otherfine-line lithography, which results in an opening having a diameter ofabout 10 nm. As shown in FIG. 9, the diameter of the opening can befurther decreased by oxidizing the silicon, thus forming a silicondioxide layer 136 over the p-type silicon layer 126 and the wallsforming opening 118. This oxidation can be formed by thermal oxidationof the silicon in an oxygen atmosphere at 800-1000.degree. C., forexample. As shown in detail in FIGS. 11 and 12, the resulting oxide hasa volume larger than the silicon consumed during the oxidation process,which further narrows the diameter of opening 118. It is desirable ifthe diameter of opening 118 can be as small as 1 nm.

Although for illustration purposes FIGS. 1, 2 and 3-9 show opening 118as being a cylindrically-shaped opening, it is preferable for opening118 to have a funnel shape as shown, for example, in FIGS. 13 and 14.This funnel-shaped opening 118 is created by performing V-groove etchingof the (100) p-type silicon layer 126 using potassium hydroxide (KOH),which results in V-shaped grooves formed along the (111) planes 138 ofthe p-type silicon 126. The V-shaped or funnel-shaped opening, as shownexplicitly in FIG. 14, facilitates movement of a DNA strand throughopening 118, and minimizes the possibility that the DNA strand willbecome balled up upon itself and thus have difficulty passing throughopening 118. Oxidation and V-groove etching can be combined to yieldeven smaller openings. Additionally, anodic oxidation can be usedinstead of thermal oxidation, as described above. Anodic oxidation hasthe additional advantage of allowing for monitoring of the opening sizeduring oxidation so that the process can be stopped when the optimumopening size is achieved.

Specifically, the opening 118 should be small enough to allow only onemolecule of the DNA strand 111 to pass through at one time.Electron-Beam lithography can yield an opening 118 as small as 10 nm,but even smaller openings are needed. Oxidation of the silicon andV-groove etching as described above can be used to further reduce theopening to the desired size of 1-2 nm. Oxidation of silicon is known toyield silicon dioxide with a volume which is about twice that of thesilicon consumed during the oxidation. Oxidation of a small opening 118will result in a gradually reduced opening size, thereby providing thedesired opening size V-groove etching of (100) oriented silicon usingKOH results in V-grooves formed by (111) planes. KOH etching through asquare SiO.sub.2 or Si.sub.3N.sub.4 mask results in a funnel shapedopening with a square cross-section. Etching through the thin siliconlayer results in an opening 118 on the other side, which is considerablesmaller in size.

Oxidation and V-groove etching can also be combined to yield evensmaller openings 118. Anodic oxidation can be used instead of thermaloxidation, which has the additional advantage of enabling the size ofthe opening 118 to be monitored during the oxidation and the oxidationcan be stopped when the appropriate size of the opening 118 is obtained.

Turning now to FIG. 15, holes 140 are etched into the silicon dioxide136 to expose n-type region 128 and n-type region 130. Metal contacts142 are then deposited onto silicon dioxide layer 136 and into holes 140to contact the respective n-type regions 128 and 130. An insulator 144is then deposited over metal contacts 142 as shown in FIG. 16, thusresulting in device 102 as shown in FIG. 1.

As further shown in FIG. 1, a portion of insulator 144 can be removed sothat leads 116 can be connected to the n-type regions 128 and 130, whichthus form the drain regions 104 and source 106, respectively. Anadditional insulator 146 is deposited over insulator 144 to seal theopenings through which leads 116 extend to contact n-type regions 128and 130. The completed device 102 can then be operated to perform theDNA sequencing as discussed above.

To identify the bases of the DNA molecule, it is desirable to measure asingle electronic charge. If the sequencing device 102 is made to have alength and width of 0.1 by 0.1.mu.m, and the thickness of the silicondioxide layer is 0.1.mu.m along the walls of the opening 118, acapacitance of 0.35 fF, a voltage variation of 0.45 mV, a devicetransconductance of 1 mS and a current variation of 0.5 nA are realized.Accordingly, a sequencing device 102 having these dimensions andcharacteristics can be used to detect a single electronic charge. Thesequencing device 102 can further be reduced in size to obtain asufficient special resolution to distinguish between differentnucleotides. The sequencing device 102 is preferably made smaller tohave an improved charge sensing capability. For example, the width ofthe sequencing device can be 10 nm, the length can be 10 nm, and theopening 118 can have a diameter of 1 nm.

Additional embodiments of the device 102 can also be fabricated. Forexample, FIG. 17 illustrates a top view of a nucleic acid sequencingdevice according to another embodiment of the present invention. In thisembodiment, the steps described above with regard to FIGS. 3 through 16are performed to form the n-type regions which ultimately form the drainand source regions. However, in this embodiment, the n-type region 128shown, for example, in FIG. 5, is formed as four separate n-typeregions, 150 in a p-type silicon layer similar to p-type silicon layer126 described above. A silicon dioxide layer 152 covers the p-typesilicon layer into which n-type regions 150 have been created. Holes 156are etched into silicon dioxide layer 152 so that metal contacts 158that are deposited on silicon dioxide layer 152 can contact n-typeregions 150. By detecting current flowing between the four drain regionsformed by n-type regions 150 and the source region (not shown), thespatial orientation of the bases on the DNA strand passing throughopening 152 can be detected.

FIG. 18 is a cross section of a nucleic acid sequencing device 160according to another embodiment of the present invention. Similar tonucleic acid sequencing device 102, 160 includes a silicon substrate162, a silicon dioxide layer 164, an n-type region 166 implanted inp-type silicon 168, and a second n-type region 170 implanted in p-typesilicon 168. Nucleic acid sequencing device 160 further has an opening172 passing therethrough. The opening can be cylindrical, or can be aV-shaped or funnel-shaped opening as described above. A silicon dioxidelayer 174 covers p-type silicon layer 168, n-type region 170 and n-typeregion 166 as shown, and decreases the diameter of opening 172 in themanner described above. An opening is etched into silicon dioxide layer172 to allow a lead 176 to be attached to n-type region 170. Anotherlead 176 is also attached to an exposed portion of n-type region 166, sothat a voltage source 178 can apply a potential across the drain region180 formed by n-type region 170 and source region 182 formed n-typeregion 166. The nucleic acid sequencing device 160 can thus be used todetect the bases of a DNA strand 182 in a manner described above.

FIG. 19 illustrates a DNA sequencing system 186 according to anotherembodiment of the present invention. System 186 includes a multi-layernucleic acid sequencing device 188 which, in this example, comprisesthree MOSFET-type devices stacked on top of each other. That is, device188 includes a silicon substrate 190 similar to silicon substrate 122described above. A silicon dioxide layer 192 is present on siliconsubstrate 190. The device 188 further includes an n-type doped siliconregion 194, a p-type silicon dioxide region 196, an n-type doped siliconregion 198, a p-type silicon dioxide region 200, an n-type doped regionsilicon region 202, a p-type silicon dioxide region 204 and an n-typedoped silicon region 206. Regions 194 through 206 are stacked on top ofeach other as shown explicitly in FIG. 19. However, as can beappreciated by one skilled in the art, the polarity of the layers can bereversed for this embodiment; and for any of the other embodimentsdiscussed herein. That is, the device 188 can comprise a p-type dopedsilicon region 194, an n-type silicon dioxide region 196, a p-type dopedsilicon region 198, and so on.

Additionally, a thin silicon dioxide layer 208 is formed over the layersas illustrated, and is also formed on the walls forming opening 210 todecrease the diameter of opening 210 in a manner described above withregard to opening 118. Also, opening 210 can be cylindrically shaped, aV-shaped groove or a funnel-shaped groove as described above. Holes areformed in silicon dioxide layer 208 so that leads 212 can be attached toregions 194, 198, 202 and 206 to couple voltage source 214, 216 and 218and current meters 220, 222 and 224 to device 188 as will now bedescribed. Voltage sources 214, 216 and 218 and current meters 220, 222and 224 are similar to voltage source 112 and current meter 114,respectively, as described above.

Specifically, leads 212 couple voltage source 214 and current meter 220in series to n-type doped silicon region 202 and n-type doped siliconregion 206. Therefore, voltage source 214 applies a voltage acrossregions 202 and 206 which are separated by p-type silicon dioxide region204. Leads 212 also couple voltage source 216 and current meter 222 ton-type doped silicon region 198 and n-type doped silicon region 202 asshown. Furthermore, leads 212 couple voltage source 218 and currentmeter 224 to n-type doped silicon region 194 and n-type doped siliconregion 202 as shown. Accordingly, as can be appreciated from FIG. 19,n-type doped silicon region 198 and n-type doped silicon region 194 actas the drain and source regions, respectively, of one MOSFET, n-typedoped silicon region 202 and n-type doped silicon region 198 act asdrain and source regions, respectively, of a second MOSFET, and n-typedoped silicon region 206 and n-type doped silicon region 202 act asdrain and source regions, respectively, of a third MOSFET. These threeMOSFET type devices can measure the current induced by the bases of aDNA strand passing through opening 210, and thus take multiplemeasurements of these bases to improve accuracy.

It is also noted that a nucleic acid sequencing device above can beconfigured to sense the bases of a nucleic acid strand without it beingnecessary for the DNA strand to pass through an opening in the devices,as shown in FIGS. 20 and 21. That is, using the techniques describedabove, a nucleic acid sequencing device 226, similar to nucleic acidsequencing device 102 shown in FIG. 1, can be fabricated having itsdrain and source regions proximate to a surface. It is noted that likecomponents shown in FIGS. 1, 20 and 21 are identified with likereference numbers. However, in place of an opening 118, one or moregrooves 228 can optionally be formed in the surface extending from thedrain region to the source region. Alternatively, no grooves are formedin the surface, but rather, the detection area for detecting nucleicacid strands 111 is present between the drain and source regions.Techniques similar to those discussed above, such as the application ofvoltage potentials by means of voltage sources 117 and 121, and creationof a pressure differential in the container 108 can be used to move thenucleic acid strands 111 in a horizontal direction along the surface ofthe device over the grooves 228. The bases in the nucleic acid strandscreate an image charge channel 230 between the drain and source regionswhich allows current to flow between the drain and source regions. Thecurrent induced in the nucleic acid sequencing device by the bases canbe measured in a manner similar to that described above.

Again, it is noted that the device 226 differs from the otherembodiments represented in FIGS. 1, 17 and 19 in that the channel 230containing the image charge is horizontal rather than vertical. Thestructure no longer contains an opening 118 as in the device 102 shownin FIGS. 1, 17 and 19, but rather this embodiment contains a chargesensitive region just above channel 230. Similar to FIG. 1, the externalelectrodes 113 and 115 are used to apply an electric field which steersthe nucleic acid strands 111 towards or away from the charge sensitiveregion. That is, the motion of the nucleic acid strands 111 iscontrolled by applying a voltage to the external electrodes 113 and 115relative to the voltage applied to the doped regions 130. Additionalelectrodes (not shown) can be added to move the nucleic acid strands 111perpendicular to the plane shown in FIG. 20.

The charge sensitive region of the device is located just above thechannel 230 and between the two doped regions 130. Identification ofindividual bases requires that the distance between the two dopedregions is on the order of a single base and that the motion of thenucleic acid strand 111 is such that each base is successively placedabove the charge sensitive region. This horizontal configuration enablesmore parallel as well as sequential analysis of the nucleic acid strands111 and does not require the fabrication of a small opening. Additionalsurface processing, such as the formation of grooves 228 as discussedabove that channel the nucleic acid strands 111 can be used to furtherenhance this approach.

The horizontal embodiment shown in FIGS. 19 and 20 is also of interestto detect the presence of a large number of nucleic acid strands 111.For instance, using an electrophoresis gel as the medium, one starts byplacing nucleic acid strands 111 of different length between theelectrodes 113 and 115. A negative voltage is applied to the electrodes113 and 115, relative to the doped regions 130. The nucleic acid strands111 will then move towards the charge sensitive regions. The smallerstrands will move faster and the larger strands will move slower. Thesmaller strands will therefore arrive first at the charge sensitiveregion followed by the larger ones. The charge accumulated in the chargesensitive region and therefore also the image charge in the channel 230therefore increases “staircase-like” with time. This results in astaircase-like increase or decrease of the current measured by currentmeter 114.

While this operation does not yield the identification of the individualbases of a single DNA/RNA strand, it does provide a measurement of thelength of the strands equivalent to the one obtained by anelectrophoresis measurement. The advantage over standard electrophoresisis that a real-time measurement of the position of the DNA/RNA strandsis obtained. In addition, the dimensions can be reduced dramaticallysince micron-sized devices can readily be made, while standardelectrophoresis uses mm if not cm-sized drift regions. This sizereduction leads to faster measurements requiring less DNA/RNA strands,while also reducing the cost of a single charge sensing device.

It is further noted that multiple DNA sensors (e.g., sequencing devices102) can be organized into a two-dimensional array 300 with electronicaddressing and readout as shown in FIG. 22. The array consists of cells302, which contain the sequencing devices 102 connected on one side toground and on the other side connected to the source, for example, of atransistor 304, so that the drain-source current of a sequencing device102 will flow into the source of its corresponding transistor 304. Thegates of the transistors 304 are connected to the word lines 306, whichin turn are connected to a decoder 308. The drain of the transistor 304in each cell 302 is connected to a sense line 310. The sense lines 310are connected to a series of sense amplifiers, shown as sense amplifier312.

The array 300 is operated by supplying an address to the decoder 308from a controller, such as a microprocessor or the like (not shown). Thedecoder 308 then applies a voltage to the word-line 306 corresponding tothe address. The sense amplifiers 312 provide the bias voltage to theselected row of sequencing devices 102. The bias voltage causes the flowof DNA molecules through the opening 118 in the selected sequencingdevice. The selected sequencing device 102 provides to its correspondingtransistor 304 a current which is proportional to the charge of theindividual nucleotides in the manner described above. The senseamplifiers measure the current of each sequencing device 102 that isselected. The array 300 thus enables multiple simultaneous measurements,which increasing the sequencing rate as compared to as single sequencingdevice 102 and also providing redundancy and additional tolerance todefective sequencing devices 102.

In addition, any of the DNA sequencers described above (e.g., sequencingdevice 102) can contain an alternative to the barrier (e.g., oxide layer136) between the semiconductor channel (e.g., channel 119 in sequencingdevice 102 shown in FIG. 1) and the medium containing the DNA molecules(e.g., liquid 110 shown in FIG. 1). For example, the oxide barrier 136can be removed, which still leaves a potential barrier between thesemiconductor and the medium. The oxide layer 136 can be replaced by awider band gap semiconductor doped with donors and/or acceptors. Theoxide layer 136 can also be replaced by an undoped wider bandgapsemiconductor layer.

Furthermore, the oxide layer 136 can be replaced with an oxidecontaining one or more silicon nanocrystals. The operation of asequencing device 102 with this type of a barrier is somewhat differentcompared than that of a sequencing device 102 with an oxide layer 136.That is, rather than directly creating an image charge in thesemiconductor channel 119, the charge of the individual nucleotidespolarizes the nanocrystal in the barrier. This polarization of thenanocrystal creates an image charge in the semiconductor channel 119.The sensitivity of the sequencing device 102 will be further enhanced aselectrons tunnel from the nucleotide into the nanocrystal. The chargeaccumulated in the nanocrystal can be removed after the measurement(e.g., current reading by current meter 114) by applying a short voltagepulse across the drain and source of the sequencing device 102.

Any of the sequencing devices described above (e.g., sequencing device102) can also be constructed without the use of a semiconductor. In thisarrangement, the middle p-type semiconductor 126 (see FIG. 1) isreplaced with an insulating layer such as silicon dioxide, while then-type source region 106 and drain region 104 are replaced by metalelectrodes. The oxide layer between the two metal electrodes must bethin enough (less than 10 nm) so that electrons can tunnel through theoxide layer. The oxide separating the two metal electrodes can be madethinner around the opening, so that tunneling only occurs at theopening.

The operation of this type of sequencing device 102-1 is described asfollows with reference to FIG. 23. As a DNA molecule passes by the thinoxide layer, it changes the local potential in the oxide and causes acurrent variation due to tunneling of electrons through the oxide layer.Since the barrier separating the molecules from the channel 119 is verythin, tunneling of electrons can occur to and from the molecules. Thistunneling can take place to/from the molecule from/to the channel 119.This current is expected to be much smaller than the current in thechannel 119 because of the large amplification within the sequencingdevice. However tunneling to and from the nanocrystal (as describedabove as an alternate barrier material) will provide a similaramplification. This tunneling will therefore provide useful andmeasurable information about the charge distribution along the DNAmolecule.

As discussed above, the size of the opening in the sequencing device(e.g., opening 118 in sequencing device 102) can be varied over a largerange. However for proper operation, the opening 118 must be smallenough so that the DNA is in close proximity to the charge sensor andlarge enough so that the DNA can traverse the opening. Since thediameter of a single stranded DNA molecule equals about 1.5 nm, theopening should be between 1 and 3 nm for optimal sensing. Largeropenings may result in reduced signal to noise ratio, but would providea larger ion flow through the opening 118.

It is further noted that certain advantages can be achieved by makingthe opening in the sequencing device asymmetric. For example, as shownin FIGS. 24 and 25, which correspond to FIGS. 13 and 14 as their relateddiscussion above, by making the initial pattern before V-groove etchingasymmetric, the final opening 118 will also be asymmetric, for example,an oval or a rectangle. Then the nucleotides, which are asymmetric, willhave a preferred orientation as they pass through the opening 118. Thisremoves the ambiguity in identifying the properties of the nucleotidesdue to their rotation around their backbone axis, and greatly simplifiesanalysis of the sensor signals. In addition, an electric field could beimposed along the longer axis of the opening 118 to align the baseintrinsic dipole moment of the nucleotide with the field. For example,the dipole moment of Cytosine is 6.44 Debye, the dipole moment ofThymine is 4.50 Debye, the dipole moment of Adenine is 2.66 Debye andthe dipole moment of Guanine is 6.88 Debye. If the field is strongenough, it can stretch the base (nucleotide) along the dipole moment,thus bringing the charges on the base nearer to the sensors increasingsensitivity. These techniques will thus make the data much easier tointerpret, and will increase the signal used to discriminate betweenbases.

In addition, as shown in FIG. 26, array 300 of the sequencing devicesdescribed above (e.g. sequencing device 102) can be used in anarrangement in which the liquid in a vessel 400 is divided into tworegions, namely, a source region 402 and one or more collection regions404. The source region 402 contains ions, fluid such as water, gel orthe other types of liquids described above, and several kinds of nucleicacid strands 406-1 and 406-2, such as DNA strands.

As shown, a collection region 404 can be formed by a collection vessel408. The collection vessel 408 can isolate the liquid in collectionregion 404 from the fluid in source region 402, in which case the fluidin source region 402 can be the same as or different than the fluid incollection region 404. Alternatively, the collection vessel 408 can beporous to allow the fluid in collection region 404 to flow into sourceregion 402 and vice-versa while being impermeable to the nucleic acidstrands 406-1 and 406-2, thus prohibiting the nucleic acid strands 406-1and 406-2 from passing through the collection vessel 408 from the sourceregion 402 into the collection region 404 and vice-versa.

The source region might contain many extraneous DNA strands plus manystrands of a particular type of DNA (type X) with a known or partiallyknown nominal sequence. Then the array 300 can be controlled asdescribed above to draw DNA strands through the openings (e.g., openings118) in the N sequencing devices 102 operating in the array 300, where Nis a number much greater than 1 (e.g., 100 or more). As DNA strandstraverse the openings 118 and are sequenced, the extraneous strands arebacked out into the source region, which is manipulated so that thisstrand is removed from the vicinity of the opening 118 it just exitedand has a negligible chance of entering another opening.

For example, strands of type X with the nominal sequence are counted,but also rejected and sent back into the source region. However, strandsof type X with one or a few differences in the base sequence detected bynano-opening j (j=1 to N) are made to traverse the openings 118 and arecaptured in a container containing a collection region of the fluid. Anysubsequent strands of type X identical to the first strand so capturedare also counted and sent to an appropriate collection container. Adifferent (or accidentally the same) non-nominal sequence of an X strandis collected at each opening 118. When sequencing stops, the ratio ofeach type of non-nominal X strand to the nominal X strands is known, anda 100% pure sample of each variant type has been collected in theindividual collection vessels 408. These pure samples then can beduplicated by PCR and studied individually. This process can be used,for example, in studying mutation and/or incorrect duplication rates,and therefore aging, in DNA from an individual.

All of the devices described above can also be modified in other ways.For example, the SiO.sub.2 oxide layer can be converted toSi.sub.3N.sub.4 in a nitrous oxide (NO) ambient for use in alkalinesolutions. Furthermore, since DNA molecules 111 are negatively charged,the molecules 111 can be attracted to the opening 118 by usingelectrodes, such as electrodes 113 and 115, to apply a positive voltageto the liquid 110 relative to the source of the device.

As discussed above, a gel can be used in place of liquid 110 to containthe DNA molecules. The use of a gel will slow down the motion of theions and further improve the signal to noise ratio. Furthermore, apressure differential can be applied across the opening to control theflow independent from the applied voltage between source and drain.

Double stranded DNA can be analyzed as well. Even though double strandedDNA is a neutral molecule, since the molecule contains charge, thenucleotides can be identified by charge sensing. In addition, othermolecules, for example, a fluorescent dye such as Hoechst dye, can beattached to single stranded DNA to enhance/modify the stiffness of themolecule thereby facilitating the insertion of the molecule into thenanometer-sized opening. Furthermore, since the above devices can byused to analyze generally any types of individual polymers, they can beused in industries dealing with polymers such as the petroleum industry,pharmaceutical industry and synthetic fiber industry, to name a few.

In addition, to facilitate the measurement of the charge of a singlemolecule (e.g., a nucleic acid strand 111 as shown in FIG. 1), a largersize particle can be attached to a single molecule. For example, a goldnanoparticle can be attached to single-stranded DNA 6. The purpose ofthe gold nanoparticle is to provide a solid anchor to the DNA molecule.The gold particle can easily be charged and discharged. As a result, thegold particle and the attached DNA molecule can be manipulated byapplying electric fields.

The gold nanoparticle attached to single stranded DNA will then beplaced in an insulating liquid such as synthetic oil, which can be usedas the liquid 110 in the arrangement shown in FIG. 1, for example. Thepurpose of the liquid is to allow the particle together with the DNAstrand to move freely. The liquid should be insulating to avoid chargeshielding by ions, which are present in conducting liquids. Syntheticoils have been identified as good candidates since they are highlyresistive and do not form chemical bonds with single stranded DNA.

The charge of the gold nanoparticles can then be measured using asemiconductor based charge sensor, such as device 102 shown in FIG. 1that includes a floating gate, or device 226 without a gate electrode asshown in FIG. 21. As charged particles approach the device, imagecharges are formed at the silicon/oxide interface in the mannerdescribed above. An appropriate bias will be applied to the device(e.g., device 102) so that it operates in the sub threshold regime,where it is most sensitive to any image charge. The induced image chargethen results in an increased conductivity of the device and is read outin the form of an increased current. As an example, it can be easilycalculated that 100 DNA molecules, which each contain 100 bases thateach carry about one third of an electronic charge, shift the thresholdvoltage of a 1.mu.m by 1.mu.m MOSFET with a 10 nm thick oxide by 150 mV.This shift can be measured by measuring the change in drain current ofthe device (e.g., as can be measured by current meter 114 shown in FIG.1). The measured charge is expected to be affected by the presence orabsence of charge-shielding cations and stray ions in DNA's hydrogenshell. Also, electric fields can be used to separate the small ions fromthe larger DNA molecules.

It is further noted that the types of nanometer-size openings describedabove, for example, opening 118 in device 102 shown in FIG. 1, can bemade as well-defined square holes as shown in FIGS. 27A and 27B. To formthese holes, a series of lines with the appropriate width and spacing isdefined in a pattern, and the pattern is then transferred into a maskingmaterial such as SiO.sub.2. The same line pattern rotated by 90.degree.is then defined and transferred into the underlying masking material sothat only the area defined by the overlapping areas between the two setsof lines is removed during etching. This process leads to a much betteredge definition of the holes compared to defining the holes in a singlelithographic step, as can be appreciated from the pattern 450 shown inFIG. 27A having openings 452. The pattern shown in FIGS. 27A and 27B wasmade with the technique described above using a line pattern with 3.mu.mwidth and 3.mu.m spacing. The resulting etch mask was then used to etchthe pits in the silicon using potassium hydroxide (KOH).

A further reduction of the line width can be achieved usingelectron-beam lithography. For example, electron-beam lithography usinga Phillips 515 scanning electron microscope (SEM) can produce a linepattern with a 100 nm width. Polymethyl methacrylite (PMMA) can be usedas an electron resist and developed with methyl iso butylketone/isopropyl alcohol (MIBK/IPA) to achieve the pattern 460 shown inFIGS. 28A and 28B having openings 462. As illustrated, the lines arewell defined and are limited by the spot size of the beam used in theelectron-beam lithography. The beginning of each line is rounded since asingle exposure with a gaussian beam has been used. This rounding can beeliminated by using the crossed line lithography technique describedwith regard to FIGS. 27A and 27B. The PMMA can also be used as an etchmask to successfully transfer the pattern into a thin SiO.sub.2 layer asshown.

Accordingly, an opening 118 can be fabricated on (100) silicon membranesby combining state-of-the-art electron beam lithography with twowell-known size reduction techniques discussed above. A scanningtransmission electron microscope (STEM) can be used to define 10 nmlines in PMMA. Crossed lines will be used to create 10 nm square holesin a SiO.sub.2 mask. KOH etching can be used to etch V-shaped pits,providing a 2-4 nm opening on the other side of the silicon membrane.The opening will be further reduced in size by thermal oxidation of thesilicon as it results in an oxide, which has about twice the volume ofthe oxidized silicon. This oxidation also provides the gate oxide, asdiscussed above.

Although only several exemplary embodiments of the present inventionhave been described in detail above, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe following claims.

We claim:
 1. A system comprising: a substrate defining a pore andincluding at least three voltage carrying regions disposed along thepore; a plurality of interconnects, each interconnect of the pluralityof interconnects uniquely connected to a voltage carrying region of theat least three voltage carrying regions; one or more power supplies inelectrical communication with the plurality of interconnects anddefining potential differences between at least two pairs of voltagecarrying regions selected from the at least three voltage carryingregions; and one or more meters to measure a difference in electricalcharacteristics between a pair of voltage carrying regions of the atleast two pairs of voltage carrying regions.
 2. The system of claim 1,wherein the electrical characteristic includes a current between thepair of voltage carrying regions.
 3. The system of claim 1, wherein thecurrent is responsive to base sequence of a nucleic acid.
 4. The systemof claim 1, further comprising two fluid volumes, the substrateseparating the two fluid volumes, the fluid volumes in fluidiccommunication via the pore.
 5. The system of claim 1, wherein thevoltage carrying region includes a doped silicon region.
 6. The systemof claim 5, wherein the doped silicon region includes an n-type dopedsilicon region.
 7. The system of claim 1, further comprising a pluralityof variable conductivity regions disposed between the at least threevoltage carrying regions.
 8. The system of claim 1, wherein the pore hasa diameter in a range of 1 nm to 10 nm.
 9. The system of claim 1,further comprising a silicon dioxide layer disposed between the at leastthree voltage carrying regions and a fluid volume within the pore.
 10. Asystem comprising: a substrate defining a pore and including at leastthree source or drain regions disposed at different positions separatedlongitudinally along the pore; a plurality of interconnects, eachinterconnect of the plurality of interconnects connected to a uniquesource or drain region of the at least three source or drain regions;one or more power supplies in electrical communication with theplurality of interconnects and defining potential differences between atleast two pairs of source or drain regions selected from the at leastsource or drain regions; and one or more current meters to measure adifference in current passing between that at least two pairs of sourceor drain regions.
 11. The system of claim 10, wherein the current isresponsive to base sequence of a nucleic acid.
 12. The system of claim10, further comprising two fluid volumes, the substrate separating thetwo fluid volumes, the fluid volumes in fluidic communication via thepore.
 13. The system of claim 10, wherein the source or drain regionincludes a doped silicon region.
 14. The system of claim 13, wherein thedoped silicon region includes an n-type doped silicon region.
 15. Thesystem of claim 10, further comprising a plurality of variableconductivity regions disposed between the at least three voltagecarrying regions.
 16. The system of claim 10, wherein the pore has adiameter in a range of 1 nm to 10 nm.
 17. The system of claim 10,further comprising a silicon dioxide layer disposed between the at leastthree voltage carrying regions and a fluid volume within the pore.
 18. Amethod of nucleic acid sequencing comprising: applying a nucleic acid toa system comprising: a substrate defining a pore and including at leastthree voltage carrying regions distributed along the pore, the nucleicacid passing through the pore; a plurality of interconnects, eachinterconnect of the plurality of interconnects connected to a uniquevoltage carrying region of the at least three voltage carrying regions;one or more power supplies in electrical communication with theplurality of interconnects and defining potential differences between atleast two pairs of voltage carrying regions selected from the at leastthree voltage carrying regions; and one or more meters to measure adifference in electrical characteristics between a pair of voltagecarrying regions of the at least two pairs of voltage carrying regions,the electrical characteristic responsive to a base sequence of thenucleic acid; and measuring the electrical characteristic.
 19. Themethod of claim 18, wherein the pore has a diameter in a range of 1 nmto 10 nm.
 20. The method of claim 18, wherein the electricalcharacteristic is current.